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Association of AMP-activated Protein Kinase Subunits With Glycogen Particles as Revealed In Situ by Immunoelectron Microscopy

Moise Bendayan, Irene Londono, Bruce E. Kemp, Grahame D. Hardie, Neil Ruderman and Marc Prentki

Department of Pathology and Cell Biology (MB,IL), Departments of Nutrition and Biochemistry (MP), and Montreal Diabetes Center (MB,MP,IL), University of Montreal, Montreal, Quebec, Canada; St. Vincent's Institute of Medical Research, University of Melbourne, Fitzroy, Victoria, Australia (BEK); Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee, Scotland (GDH); and Diabetes Research Unit, University of Boston, Boston, Massachusetts (NR)

Correspondence to: Doctor M. Bendayan, Department of Pathology and Cell Biology, Faculty of Medicine, University of Montreal, 2900 Edouard MontPetit C.P. 6128 Succ. Centre Ville, Montreal, Quebec H3C 3J7, Canada. E-mail: moise.bendayan{at}umontreal.ca

Immunogold cytochemistry was applied to reveal the intracellular location of AMP-activated protein kinase (AMPK) subunits in liver tissue of normal rats fed ad libitum. AMPK and β subunits were located both in the cytosol and in close association with rosettes of glycogen particles ( particles). To reveal their true in situ association with glycogen, particular tissue processing conditions that retain glycogen in the cells were required. These included fixation with a combination of glutaraldehyde and paraformaldehyde, followed by postfixation with osmium tetroxide and lead citrate and embedding in Epon. Processing by less-stringent fixation conditions and embedding in Lowicryl led to the extraction of the glycogen deposits, which in turn resulted in the absence of any labeling. This indicates that the loss of glycogen deposits leads to the loss of closely associated proteins. Labeling for the ₁ and ₂ subunits of AMPK was found to be about 2-fold greater over glycogen than over cytosol, whereas labeling for β₁ was 8-fold higher over the glycogen particles than over the cytosol. Immunogold combined with morphometric analysis demonstrated that the β₁ subunits are located at the periphery of the glycogen rosettes, consistent with a recent hypothesis developed via biochemical approaches. (J Histochem Cytochem 57:963–971, 2009)

Key Words: AMP-activated kinase • glycogen • immunocytochemistry • protein A–gold • liver tissue

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